PROJECT SUMMARY Zika virus (ZIKV) is a flavivirus that is transmitted to its human host principally by mosquitos. Most individuals infected with ZIKV remain asymptomatic. In rare cases, ZIKV infection may be associated with Guillain-Barre syndrome and, in even rarer cases, death. However, infections in pregnant women can result in the transplacental transmission of ZIKV to the fetus, leading to microcephaly, as observed in the recent outbreaks in South and Central America. There is currently no antiviral treatment or vaccine for ZIKV, and developing an understanding of the pathogenesis of ZIKV infections is, thus, of extremely high priority. We have recently identified humans with deficiencies of either ISG15 or USP18, two important downregulators of the type I IFN signaling pathway. Our preliminary data have revealed that ISG15- and USP18-deficient cell lines are more resistant to ZIKV than wild-type (WT) cell lines. Our preliminary data for cells derived from ISG15-deficient individuals have shown that only a handful of interferon-stimulated genes (ISGs) are involved in controlling ZIKV infection. In the work described in this proposal, we will refine the list of ISGs involved in the control of ZIKV infection, by comparing ISG15- and USP18-deficient cells with WT cells. This should make it possible to further narrow down the number of ISGs potentially involved in the anti-ZIKV response from more than 400 to <20. We will identify and characterize the principal factors restricting ZIKV infection, at the RNA (Aim 1) and protein (Aim 2) levels. We will then assess the ability of seven ZIKV strains to induce type I IFN production in different cell types, and their sensitivity to treatment with type I IFN. By improving our understanding of the human determinants of ZIKV resistance, we should be able to develop new hypotheses concerning both human susceptibility in vivo and drug development.